I have some problems in GAP( Gtpase activating protein) assay. First, I
describe the assay system that I use for GAP. Smg( small G protein) was
pre-formed GTP form, add the GTP form smg into cell lysate and incubate for
proper time, the reaction was stopped by adding SDS/EDTA solution, the
stopped mixture was developed by TLC and obtain GTP/GTP+GDP ratio as GTPase
activity. Because I have no smg antibody, I discarded the
immunoprecipitation step before stop reaction.
Problem 1. Is immunoprecipitation a essential step? Because
immunoprecipitation takes time and seemed to affect estimation of GTPase
activity. Another problem that I concerned is that immunoprecipitation
cause to get lower lower radioactivity because some guanosine nucleotide
exchange factor cause radioactive bound-form guanosine nucleotides turned
into free form because of replacement of cold free guanosine nuceleotides.
Problem 2. If I want to measure GAP activity change during stimulation(
such as high potassium), how can I obtain the stimulated cell lysates at
different time points?
If you know how to resolve these problems, please tell me. Thank you!